1
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Zhou D, Zeng Y, Luo W, Leng C, Li C. Senior-Loken Syndrome: Ocular Perspectives on Genetics, Pathogenesis, and Management. Biomolecules 2025; 15:667. [PMID: 40427560 PMCID: PMC12109206 DOI: 10.3390/biom15050667] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2025] [Revised: 05/02/2025] [Accepted: 05/03/2025] [Indexed: 05/29/2025] Open
Abstract
Senior-Loken syndrome (SLSN) is a group of rare autosomal recessive disorders caused by dysfunction of the primary cilium, primarily affecting the kidneys (typically leading to nephronophthisis) and eyes (typically leading to retinal degeneration). Moreover, patients with SLSN may experience additional multisystemic symptoms, such as developmental delay, intellectual disability, ataxia, and nystagmus. To date, eight genes have been demonstrated to cause SLSN, all encoding for proteins involved in the structure and functions of the primary cilium. This places SLSN within an expanding category of diseases known as "ciliopathies". Due to the genetic heterogeneity and significant phenotypic overlap with other ciliopathies, establishing a definitive diagnosis during the initial consultation remains a challenge for clinicians. Furthermore, current research on SLSN-related ciliopathies predominantly focuses on renal involvement, while the ocular manifestations remain insufficiently explored and lack a comprehensive review. Therefore, with the goal of offering practical guidance for clinical practice, this review aims to provide a comprehensive overview of the clinical features, along with an ocular perspective on the molecular mechanisms, genetic underpinnings, and advances in the treatment of SLSN.
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Affiliation(s)
- Di Zhou
- Center for Drug Evaluation, National Medical Products Administration, Beijing 100076, China;
| | - Yi Zeng
- Xiangya School of Medicine, Central South University, Changsha 410013, China; (Y.Z.); (W.L.)
| | - Weihan Luo
- Xiangya School of Medicine, Central South University, Changsha 410013, China; (Y.Z.); (W.L.)
| | - Chenyang Leng
- Xiangya School of Medicine, Central South University, Changsha 410013, China; (Y.Z.); (W.L.)
| | - Chen Li
- Xiangya School of Medicine, Central South University, Changsha 410013, China; (Y.Z.); (W.L.)
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2
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Zhang P, Xu Z. The advancements in precision medicine for Leber congenital amaurosis: Breakthroughs from genetic diagnosis to therapy. Surv Ophthalmol 2025:S0039-6257(25)00070-0. [PMID: 40311816 DOI: 10.1016/j.survophthal.2025.04.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 04/15/2025] [Accepted: 04/28/2025] [Indexed: 05/03/2025]
Abstract
Leber congenital amaurosis (LCA) is a hereditary retinal disease, typically manifesting as severe vision impairment in infancy. With the advancement of precision medicine, genetic diagnosis and targeted therapies offer new hope for LCA patients, significantly improving both diagnostic accuracy and therapeutic efficacy. We summarize the epidemiological characteristics, clinical manifestations, and molecular genetics underlying LCA. It also highlights recent developments in precision treatment strategies, including gene replacement therapy, CRISPR/Cas9-mediated gene editing, and antisense oligonucleotide therapies. In addition, we discuss the applications of induced pluripotent stem cells and retinal organoids in LCA treatment research. Furthermore, we explore preventive strategies and future treatment directions for LCA, including the development of novel gene therapy vectors, the optimization of combinatorial treatment strategies, and the formulation of personalized treatment approaches. These advancements hold significant potential to offer improved treatment options and enhance the quality of life for LCA patients.
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Affiliation(s)
- Pei Zhang
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
| | - Zhuping Xu
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
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3
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Chen KY, Chan HC, Chan CM. Can Stem Cell Therapy Revolutionize Ocular Disease Treatment? A Critical Review of Preclinical and Clinical Advances. Stem Cell Rev Rep 2025:10.1007/s12015-025-10884-x. [PMID: 40266467 DOI: 10.1007/s12015-025-10884-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/08/2025] [Indexed: 04/24/2025]
Abstract
Stem cell therapy in regenerative medicine has a scope for treating ocular diseases. Stem cell therapy aims to repair damaged tissue and restore vision. The present review focuses on the advancements in stem cell therapies for ocular disorders, their mechanism of action, and clinical applications while addressing some outstanding challenges. Stem cells that include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells have regenerative potential for ocular repair. They differentiate into specialized ocular cell types, conduct neuroprotection, and modulate immune responses. It is emphasized in preclinical and clinical studies that stem cell therapy can treat corneal disorders such as limbal stem cell deficiency, retinal diseases like dry age macular degeneration and retinitis pigmentosa, and diabetic retinopathy. Various studies suggested that stem cells have considerable scope in glaucoma treatment by supporting retinal ganglion cell survival and optic nerve regeneration. Advanced approaches such as gene editing, organoid generation, and artificial intelligence enhance these therapies. Effective delivery to target areas, engraftment, orientation, and long-term survival of transplanted cells need optimization. Issues such as immune rejection and tumorigenicity must be addressed. This approach is further hindered by regulatory issues and overly complicated approval processes and trials. Ethical issues related to sourcing embryonic stem cells and patient consent complicate the issue. The cost of manufacturing stem cells and their accessibility are other factors posing potential barriers to widespread application. These regulatory, ethical, and economic issues must be tackled if stem cell treatments are to be made safe, accessible, and effective. Future studies will include refining therapeutic protocols, scaling manufacturing processes, and overcoming socio-economic barriers, eventually improving clinical outcomes.
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Affiliation(s)
- Kai-Yang Chen
- School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Hoi-Chun Chan
- School of Pharmacy, China Medical University, Taichung, Taiwan
| | - Chi-Ming Chan
- Department of Ophthalmology, Cardinal Tien Hospital, New Taipei City, Taiwan.
- School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.
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4
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Tachida Y, Manian KV, Butcher R, Levy JM, Pendse N, Hennessey E, Liu DR, Pierce EA, Liu Q, Comander J. Systematic empirical evaluation of individual base editing targets: Validating therapeutic targets in USH2A and comparison of methods. Mol Ther 2025; 33:1466-1484. [PMID: 39881543 PMCID: PMC11997516 DOI: 10.1016/j.ymthe.2025.01.042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2024] [Revised: 11/22/2024] [Accepted: 01/24/2025] [Indexed: 01/31/2025] Open
Abstract
Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids vs. transgenes) and assays (next-generation sequencing vs. Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knockin mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955∗). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65% ± 3% at the mutant base pair and of 52% ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.
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Affiliation(s)
- Yuki Tachida
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Kannan V Manian
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Rossano Butcher
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Jonathan M Levy
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02142, USA
| | - Nachiket Pendse
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Erin Hennessey
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - David R Liu
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02142, USA
| | - Eric A Pierce
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Qin Liu
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA
| | - Jason Comander
- Ocular Genomics Institute, Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA.
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5
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Gu B, Li M, Li D, Huang K. CRISPR-Cas9 Targeting PCSK9: A Promising Therapeutic Approach for Atherosclerosis. J Cardiovasc Transl Res 2025; 18:424-441. [PMID: 39804565 DOI: 10.1007/s12265-024-10587-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2024] [Accepted: 12/28/2024] [Indexed: 05/01/2025]
Abstract
CRISPR-Cas9 gene editing technology, as an innovative biomedical tool, holds significant potential in the prevention and treatment of atherosclerosis. By precisely editing key genes such as PCSK9, CRISPR-Cas9 offers the possibility of long-term regulation of low-density lipoprotein cholesterol (LDL-C), which may reduce the risk of cardiovascular diseases. Early clinical studies of gene editing therapies like VERVE-101 have yielded encouraging results, highlighting both the feasibility and potential efficacy of this technology. However, clinical applications still face challenges such as off-target effects, immunogenicity, and long-term safety. Future research should focus on enhancing the specificity and efficiency of gene editing, optimizing delivery systems, and improving personalized treatment strategies. Additionally, the establishment of ethical and legal regulatory frameworks will be critical for the safe adoption of this technology. With the continued advancement of gene editing technology, CRISPR-Cas9 may become an important tool for treating atherosclerosis and other complex diseases.
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Affiliation(s)
- Bin Gu
- Department of Cardiology, Affiliated Hospital of Southwest Medical University, No.1 Section 1, Xiang Lin Road, Longmatan District, Luzhou, Sichuan, 646000, China
| | - Min Li
- Department of Cardiology, Neijiang Dongxing District People's Hospital, Neijiang, Sichuan, 641300, China
| | - Dan Li
- Department of Cardiology, Neijiang Dongxing District People's Hospital, Neijiang, Sichuan, 641300, China
| | - Kaisen Huang
- Department of Cardiology, Affiliated Hospital of Southwest Medical University, No.1 Section 1, Xiang Lin Road, Longmatan District, Luzhou, Sichuan, 646000, China.
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6
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Cetin B, Erendor F, Eksi YE, Sanlioglu AD, Sanlioglu S. Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects. Expert Rev Mol Med 2025; 27:e16. [PMID: 40160040 DOI: 10.1017/erm.2025.10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Genome editing has recently evolved from a theoretical concept to a powerful and versatile set of tools. The discovery and implementation of CRISPR-Cas9 technology have propelled the field further into a new era. This RNA-guided system allows for specific modification of target genes, offering high accuracy and efficiency. Encouraging results are being announced in clinical trials employed in conditions like sickle cell disease (SCD) and transfusion-dependent beta-thalassaemia (TDT). The path finally led the way to the recent FDA approval of the first gene therapy drug utilising the CRISPR/Cas9 system to edit autologous CD34+ haematopoietic stem cells in SCD patients (Casgevy). Ongoing research explores the potential of CRISPR technology for cancer therapies, HIV treatment and other complex diseases. Despite its remarkable potential, CRISPR technology faces challenges such as off-target effects, suboptimal delivery systems, long-term safety concerns, scalability, ethical dilemmas and potential repercussions of genetic alterations, particularly in the case of germline editing. Here, we examine the transformative role of CRISPR technologies, including base editing and prime editing approaches, in modifying the genetic and epigenetic codes in the human genome and provide a comprehensive focus, particularly on relevant clinical applications, to unlock the full potential and challenges of gene editing.
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Affiliation(s)
- Busra Cetin
- Department of Gene and Cell Therapy, Faculty of Medicine, Akdeniz University, Antalya, Turkey
| | - Fulya Erendor
- Department of Gene and Cell Therapy, Faculty of Medicine, Akdeniz University, Antalya, Turkey
| | - Yunus Emre Eksi
- Department of Gene and Cell Therapy, Faculty of Medicine, Akdeniz University, Antalya, Turkey
| | - Ahter D Sanlioglu
- Department of Gene and Cell Therapy, Faculty of Medicine, Akdeniz University, Antalya, Turkey
| | - Salih Sanlioglu
- Department of Gene and Cell Therapy, Faculty of Medicine, Akdeniz University, Antalya, Turkey
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7
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Liu D, Cao D, Han R. Recent advances in therapeutic gene-editing technologies. Mol Ther 2025:S1525-0016(25)00200-X. [PMID: 40119516 DOI: 10.1016/j.ymthe.2025.03.026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Revised: 02/26/2025] [Accepted: 03/17/2025] [Indexed: 03/24/2025] Open
Abstract
The advent of gene-editing technologies, particularly CRISPR-based systems, has revolutionized the landscape of biomedical research and gene therapy. Ongoing research in gene editing has led to the rapid iteration of CRISPR technologies, such as base and prime editors, enabling precise nucleotide changes without the need for generating harmful double-strand breaks (DSBs). Furthermore, innovations such as CRISPR fusion systems with DNA recombinases, DNA polymerases, and DNA ligases have expanded the size limitations for edited sequences, opening new avenues for therapeutic development. Beyond the CRISPR system, mobile genetic elements (MGEs) and epigenetic editors are emerging as efficient alternatives for precise large insertions or stable gene manipulation in mammalian cells. These advances collectively set the stage for next-generation gene therapy development. This review highlights recent developments of genetic and epigenetic editing tools and explores preclinical innovations poised to advance the field.
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Affiliation(s)
- Dongqi Liu
- Department of Pediatrics, Department of Molecular and Medical Genetics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Di Cao
- Department of Pediatrics, Department of Molecular and Medical Genetics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Renzhi Han
- Department of Pediatrics, Department of Molecular and Medical Genetics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
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8
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Lee H, Rho WY, Kim YH, Chang H, Jun BH. CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations. Molecules 2025; 30:542. [PMID: 39942646 PMCID: PMC11820414 DOI: 10.3390/molecules30030542] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2024] [Revised: 01/07/2025] [Accepted: 01/17/2025] [Indexed: 02/16/2025] Open
Abstract
The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9's clinical efficacy, positioning them as pivotal tools for future gene-editing therapies.
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Affiliation(s)
- Hyunwoo Lee
- Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea; (H.L.); (Y.-H.K.)
| | - Won-Yeop Rho
- School of International Engineering and Science, Jeonbuk National University, Jeonju 54896, Republic of Korea;
| | - Yoon-Hee Kim
- Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea; (H.L.); (Y.-H.K.)
| | - Hyejin Chang
- Division of Science Education, Kangwon National University, 1 Gangwondaehakgil, Chuncheon-si 24341, Republic of Korea
| | - Bong-Hyun Jun
- Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea; (H.L.); (Y.-H.K.)
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9
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Nieland L, Vrijmoet AB, Jetten IW, Rufino-Ramos D, de Reus AJEM, Breyne K, Kleinstiver BP, Maguire CA, Broekman MLD, Breakefield XO, Abels ER. CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice. Mol Ther 2025; 33:133-151. [PMID: 39563028 PMCID: PMC11764731 DOI: 10.1016/j.ymthe.2024.11.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2024] [Revised: 09/23/2024] [Accepted: 11/15/2024] [Indexed: 11/21/2024] Open
Abstract
Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice.
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Affiliation(s)
- Lisa Nieland
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA; Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands
| | - Anne B Vrijmoet
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA
| | - Isabelle W Jetten
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA
| | - David Rufino-Ramos
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
| | - Alexandra J E M de Reus
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA
| | - Koen Breyne
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA
| | - Benjamin P Kleinstiver
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
| | - Casey A Maguire
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA; Department of Neurology, Massachusetts General Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02116, USA
| | - Marike L D Broekman
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA; Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; Department of Neurosurgery, Haaglanden Medical Center, 2512 VA The Hague, the Netherlands; Department of Neurosurgery, Leiden University Medical Center, 2300 RC Leiden, the Netherlands
| | - Xandra O Breakefield
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA
| | - Erik R Abels
- Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands.
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10
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Bansal M. Advances in retina genetics: Progress, potential, and challenges. Indian J Ophthalmol 2025; 73:S31-S36. [PMID: 39257094 PMCID: PMC11834934 DOI: 10.4103/ijo.ijo_3334_23] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2023] [Revised: 03/05/2024] [Accepted: 07/02/2024] [Indexed: 09/12/2024] Open
Abstract
The field of retinal genetics has seen remarkable advancements lately, reshaping our understanding of various retinal conditions, including age-related macular degeneration, diabetic retinopathy, and inherited retinal dystrophies. The purpose of this review is to provide an overview of the current status of genetics in the retina, covering the progress made, the expected future developments, and the challenges yet to be overcome. We highlight key advancements such as the advent of next-generation sequencing, which has exponentially enhanced the discovery of genetic mutations, thus also enabling personalized medicine/therapeutic approaches. Stem cells, gene augmentation, and gene-editing techniques such as CRISPR/Cas9 are discussed, in which we highlight ongoing research as well as their potential in the targeted treatment of retinal diseases. Despite these promising advancements, the field faces significant challenges, such as the complex interpretation of genetic data, ethical considerations, and the translational gap from bench to bedside. This review serves as a comprehensive guide not only to ophthalmologists but also to other healthcare professionals, scientists, and policymakers, providing insights into the rapidly evolving landscape of retinal genetics. It aims to stimulate further research and collaboration to surmount existing challenges and harness the full potential of genetic advancements for retinal health.
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Affiliation(s)
- Mayank Bansal
- Vitreo-Retinal Surgery, CSIR - Institute of Genomics and Integrative Biology, Sightgenics Research and Fortis Memorial Research Institute, Delhi, India
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11
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Dua PH, Simon BMJ, Marley CB, Feliciano CM, Watry HL, Steury D, Abraham A, Gilbertson EN, Ramey GD, Capra JA, Conklin BR, Judge LM. Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.20.629813. [PMID: 39763989 PMCID: PMC11702708 DOI: 10.1101/2024.12.20.629813] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/15/2025]
Abstract
Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplo-sufficient. We previously edited a dominant NEFL missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause disease. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach for two different missense mutations and demonstrated its therapeutic potential in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent byproduct of excision editing, failed to reliably disrupt mutant allele expression. We deployed alternative strategies and novel molecular assays to increase therapeutic editing outcomes while maintaining specificity for the mutant allele. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.
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Affiliation(s)
- Poorvi H. Dua
- Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA
- Gladstone Institutes, San Francisco, CA, United States
| | | | - Chiara B.E. Marley
- Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA
- Gladstone Institutes, San Francisco, CA, United States
| | - Carissa M. Feliciano
- Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA
- Gladstone Institutes, San Francisco, CA, United States
| | | | - Dylan Steury
- Gladstone Institutes, San Francisco, CA, United States
- University of California, Berkeley, Berkeley, CA, USA
| | - Abin Abraham
- Vanderbilt Genetics Institute, Vanderbilt University, Nashville, TN, USA
- Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN, USA
- Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA
| | - Erin N. Gilbertson
- Biomedical Informatics Graduate Program, University of California, San Francisco, San Francisco, CA, USA
- Bakar Computational Health Sciences Institute, University of California, San Francisco, CA, USA
- Department of Genetics, Yale School of Medicine, New Haven, CT, USA
| | - Grace D. Ramey
- Biomedical Informatics Graduate Program, University of California, San Francisco, San Francisco, CA, USA
- Bakar Computational Health Sciences Institute, University of California, San Francisco, CA, USA
| | - John A. Capra
- Bakar Computational Health Sciences Institute, University of California, San Francisco, CA, USA
- Department of Epidemiology and Biostatistics, University of California, San Francisco, CA
| | - Bruce R. Conklin
- Gladstone Institutes, San Francisco, CA, United States
- Department of Ophthalmology, University of California, San Francisco, San Francisco, CA, United States
- Department of Medicine, University of California, San Francisco, San Francisco, CA, United States
- Innovative Genomics Institute, Berkeley, CA, United States
| | - Luke M. Judge
- Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA
- Gladstone Institutes, San Francisco, CA, United States
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12
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Wang JH, Zhan W, Gallagher TL, Gao G. Recombinant adeno-associated virus as a delivery platform for ocular gene therapy: A comprehensive review. Mol Ther 2024; 32:4185-4207. [PMID: 39489915 PMCID: PMC11638839 DOI: 10.1016/j.ymthe.2024.10.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Revised: 09/18/2024] [Accepted: 10/23/2024] [Indexed: 11/05/2024] Open
Abstract
Adeno-associated virus (AAV) has emerged as a leading platform for in vivo gene therapy, particularly in ocular diseases. AAV-based therapies are characterized by low pathogenicity and broad tissue tropism and have demonstrated clinical success, as exemplified by voretigene neparvovec-rzyl (Luxturna) being the first gene therapy to be approved by the U.S. Food and Drug Administration to treat RPE65-associated Leber congenital amaurosis (LCA). However, several challenges remain in the development of AAV-based gene therapies, including immune responses, limited cargo capacity, and the need for enhanced transduction efficiency, especially for intravitreal delivery to photoreceptors and retinal pigment epithelium cells. This review explores the biology of AAVs in the context of gene therapy, innovations in capsid engineering, and clinical advancements in AAV-based ocular gene therapy. We highlight ongoing clinical trials targeting inherited retinal diseases and acquired conditions, discuss immune-related limitations, and examine novel strategies for enhancing AAV vector performance to address current barriers.
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Affiliation(s)
- Jiang-Hui Wang
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC 3002, Australia
| | - Wei Zhan
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | - Thomas L Gallagher
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA
| | - Guangping Gao
- Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA; Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA 01605, USA.
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13
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D’Esposito F, Zeppieri M, Cordeiro MF, Capobianco M, Avitabile A, Gagliano G, Musa M, Barboni P, Gagliano C. Insights on the Genetic and Phenotypic Complexities of Optic Neuropathies. Genes (Basel) 2024; 15:1559. [PMID: 39766826 PMCID: PMC11675667 DOI: 10.3390/genes15121559] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Revised: 11/27/2024] [Accepted: 11/28/2024] [Indexed: 01/11/2025] Open
Abstract
Background/Objectives: Optic neuropathies are a category of illnesses that ultimately cause damage to the optic nerve, leading to vision impairment and possible blindness. Disorders such as dominant optic atrophy (DOA), Leber hereditary optic neuropathy (LHON), and glaucoma demonstrate intricate genetic foundations and varied phenotypic manifestations. This narrative review study seeks to consolidate existing knowledge on the genetic and molecular mechanisms underlying ocular neuropathies, examine genotype-phenotype correlations, and assess novel therapeutic options to improve diagnostic and treatment methodologies. Methods: A systematic literature review was performed in October 2024, utilizing PubMed, Medline, the Cochrane Library, and ClinicalTrials.gov. Search terms encompassed "optic neuropathy", "genetic variants", "LHON", "DOA", "glaucoma", and "molecular therapies". Studies were chosen according to established inclusion criteria, concentrating on the genetic and molecular dimensions of optic neuropathies and their therapeutic ramifications. Results: The results indicate that DOA and LHON are mostly associated with the mitochondrial dysfunction resulting from pathogenic variants in nuclear genes, mainly OPA1, and mitochondrial DNA (mtDNA) genes, respectively. Glaucoma, especially its intricate variants, is linked to variants in genes like MYOC, OPTN, and TBK1. Molecular mechanisms, such as oxidative stress and inflammatory modulation, are pivotal in disease progression. Innovative therapeutics, including gene therapy, RNA-based treatments, and antioxidants such as idebenone, exhibit promise for alleviating optic nerve damage and safeguarding vision. Conclusions: Genetic and molecular investigations have markedly enhanced our comprehension of ocular neuropathies. The amalgamation of genetic and phenotypic data is essential for customized medical strategies. Additional research is required to enhance therapeutic strategies and fill the gaps in our understanding of the underlying pathophysiology. This interdisciplinary approach shows potential for enhancing patient outcomes in ocular neuropathies.
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MESH Headings
- Humans
- Optic Atrophy, Hereditary, Leber/genetics
- Optic Atrophy, Hereditary, Leber/therapy
- Optic Atrophy, Hereditary, Leber/pathology
- Phenotype
- Glaucoma/genetics
- Glaucoma/therapy
- Glaucoma/pathology
- Optic Nerve Diseases/genetics
- Optic Atrophy, Autosomal Dominant/genetics
- Optic Atrophy, Autosomal Dominant/therapy
- Optic Atrophy, Autosomal Dominant/pathology
- DNA, Mitochondrial/genetics
- Genetic Association Studies
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Affiliation(s)
- Fabiana D’Esposito
- Imperial College Ophthalmic Research Group (ICORG) Unit, Imperial College, London NW1 5QH, UK; (F.D.)
- Eye Clinic, Department of Neurosciences, Reproductive Sciences and Dentistry, University of Naples Federico II, 80131 Naples, Italy
| | - Marco Zeppieri
- Department of Ophthalmology, University Hospital of Udine, Piazzale Santa Maria della Misericordia 15, 33100 Udine, Italy
| | - Maria Francesca Cordeiro
- Imperial College Ophthalmic Research Group (ICORG) Unit, Imperial College, London NW1 5QH, UK; (F.D.)
- Western Eye Hospital, Imperial College Healthcare NHS Trust, London NW1 5QH, UK
| | - Matteo Capobianco
- Eye Clinic, Catania University San Marco Hospital, Viale Carlo Azeglio Ciampi, 95121 Catania, Italy
| | - Alessandro Avitabile
- Eye Clinic, Catania University San Marco Hospital, Viale Carlo Azeglio Ciampi, 95121 Catania, Italy
| | - Giuseppe Gagliano
- Eye Clinic, Catania University San Marco Hospital, Viale Carlo Azeglio Ciampi, 95121 Catania, Italy
| | - Mutali Musa
- Department of Optometry, University of Benin, Benin City 300238, Nigeria
| | - Piero Barboni
- Department of Ophthalmology, University Vita-Salute, IRCCS Ospedale San Raffaele, 20132 Milan, Italy
- Studio Oculistico d’Azeglio, 40123 Bologna, Italy
| | - Caterina Gagliano
- Department of Medicine and Surgery, University of Enna “Kore”, Piazza dell’Università, 94100 Enna, Italy
- Mediterranean Foundation “G.B. Morgagni”, 95125 Catania, Italy
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14
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Gong X, Hertle RW. Infantile Nystagmus Syndrome-Associated Inherited Retinal Diseases: Perspectives from Gene Therapy Clinical Trials. Life (Basel) 2024; 14:1356. [PMID: 39598155 PMCID: PMC11595273 DOI: 10.3390/life14111356] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Revised: 09/27/2024] [Accepted: 10/21/2024] [Indexed: 11/29/2024] Open
Abstract
Inherited retinal diseases (IRDs) are a clinically and genetically diverse group of progressive degenerative disorders that can result in severe visual impairment or complete blindness. Despite their predominantly monogenic inheritance patterns, the genetic complexity of over 300 identified disease-causing genes presents a significant challenge in correlating clinical phenotypes with genotypes. Achieving a molecular diagnosis is crucial for providing patients with definitive diagnostic clarity and facilitating access to emerging gene-based therapies and ongoing clinical trials. Recent advances in next-generation sequencing technologies have markedly enhanced our ability to identify genes and genetic defects leading to IRDs, thereby propelling the development of gene-based therapies. The clinical success of voretigene neparvovec (Luxturna), the first approved retinal gene therapy for RPE65-associated Leber congenital amaurosis (LCA), has spurred considerable research and development in gene-based therapies, highlighting the importance of reviewing the current status of gene therapy for IRDs, particularly those utilizing adeno-associated virus (AAV)-based therapies. As novel disease-causing mutations continue to be discovered and more targeted gene therapies are developed, integrating these treatment opportunities into the standard care for IRD patients becomes increasingly critical. This review provides an update on the diverse phenotypic-genotypic landscape of IRDs, with a specific focus on recent advances in the understanding of IRDs in children with infantile nystagmus syndrome (INS). We highlight the complexities of the genotypic-phenotypic landscape of INS-associated IRDs, including conditions such as achromatopsia, LCA, congenital stationary night blindness, and subtypes of retinitis pigmentosa. Additionally, we provide an updated overview of AAV-based gene therapies for these diseases and discuss the potential of gene-based therapies for underlying IRDs that lead to INS, offering a valuable resource for pediatric patients potentially eligible for ongoing clinical trials.
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Affiliation(s)
- Xiaoming Gong
- Department of Ophthalmology, Akron Children’s Hospital, Akron, OH 44308, USA;
- Vision Center of Excellence, Rebecca D. Considine Research Institute, Akron Children’s Hospital, Akron, OH 44308, USA
| | - Richard W. Hertle
- Department of Ophthalmology, Akron Children’s Hospital, Akron, OH 44308, USA;
- Vision Center of Excellence, Rebecca D. Considine Research Institute, Akron Children’s Hospital, Akron, OH 44308, USA
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15
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Wang T, Yu T, Liu Q, Sung TC, Higuchi A. Lipid nanoparticle technology-mediated therapeutic gene manipulation in the eyes. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102236. [PMID: 39005878 PMCID: PMC11245926 DOI: 10.1016/j.omtn.2024.102236] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 07/16/2024]
Abstract
Millions of people worldwide have hereditary genetic disorders, trauma, infectious diseases, or cancer of the eyes, and many of these eye diseases lead to irreversible blindness, which is a major public health burden. The eye is a relatively small and immune-privileged organ. The use of nucleic acid-based drugs to manipulate malfunctioning genes that target the root of ocular diseases is regarded as a therapeutic approach with great promise. However, there are still some challenges for utilizing nucleic acid therapeutics in vivo because of certain unfavorable characteristics, such as instability, biological carrier-dependent cellular uptake, short pharmacokinetic profiles in vivo (RNA), and on-target and off-target side effects (DNA). The development of lipid nanoparticles (LNPs) as gene vehicles is revolutionary progress that has contributed the clinical application of nucleic acid therapeutics. LNPs have the capability to entrap and transport various genetic materials such as small interfering RNA, mRNA, DNA, and gene editing complexes. This opens up avenues for addressing ocular diseases through the suppression of pathogenic genes, the expression of therapeutic proteins, or the correction of genetic defects. Here, we delve into the cutting-edge LNP technology for ocular gene therapy, encompassing formulation designs, preclinical development, and clinical translation.
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Affiliation(s)
- Ting Wang
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China
| | - Tao Yu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China
| | - Qian Liu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China
| | - Tzu-Cheng Sung
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China
| | - Akon Higuchi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, No. 270, Xueyuan Road, Wenzhou, Zhejiang 325027, China
- Department of Chemical and Materials Engineering, National Central University, No. 300, Jhongda RD, Jhongli, Taoyuan 32001, Taiwan
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16
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Huang XX, Wang YM, Xie MY, Sun YQ, Zhao XH, Chen YH, Chen JQ, Han SY, Zhou MW, Sun XD. Publication trends of Leber congenital amaurosis researches: a bibliometric study during 2002-2022. Int J Ophthalmol 2024; 17:1501-1509. [PMID: 39156783 PMCID: PMC11286431 DOI: 10.18240/ijo.2024.08.17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2024] [Accepted: 04/12/2024] [Indexed: 08/20/2024] Open
Abstract
AIM To analyze the changes in scientific output relating to Leber congenital amaurosis (LCA) and forecast the study trends in this field. METHODS All of the publications in the field of LCA from 2002 to 2022 were collected from Web of Science (WOS) database. We analyzed the quantity (number of publications), quality (citation and H-index) and development trends (relative research interest, RRI) of published LCA research over the last two decades. Moreover, VOSviewer software was applied to define the co-occurrence network of keywords in this field. RESULTS A total of 2158 publications were ultimately examined. We found that the focus on LCA kept rising and peaked in 2015 and 2018, which is consistent with the development trend of gene therapy. The USA has contributed most to this field with 1162 publications, 56 674 citations and the highest H-index value (116). The keywords analysis was divided into five clusters to show the hotspots in the field of LCA, namely mechanism-related, genotype-related, local phenotype-related, system phenotype-related, and therapy-related. We also identified gene therapy and anti-retinal degeneration therapy as a major focus in recent years. CONCLUSION Our study illustrates historical research process and future development trends in LCA field. This may help to guide the orientation for further clinical diagnosis, treatment and scientific research.
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Affiliation(s)
- Xiao-Xu Huang
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Yi-Min Wang
- Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai 200030, China
| | - Min-Yue Xie
- Beijing Tongren Hospital, Capital Medical University, Beijing 100054, China
| | - Yi-Qing Sun
- Eberly College of Science, Penn State University, University Park 16802-1503, United States
| | - Xiao-Huan Zhao
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Yu-Hong Chen
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Jie-Qiong Chen
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Si-Yang Han
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Min-Wen Zhou
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
| | - Xiao-Dong Sun
- Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China
- National Clinical Research Center for Eye Disease, Shanghai 200080, China
- Shanghai Key Laboratory of Ocular Fundus Diseases, Shanghai 200080, China
- Shanghai Engineering Center for Visual Science and Photomedicine, Shanghai 200080, China
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17
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Gowda DAA, Birappa G, Rajkumar S, Ajaykumar CB, Srikanth B, Kim SL, Singh V, Jayachandran A, Lee J, Ramakrishna S. Recent progress in CRISPR/Cas9 system for eye disorders. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2024; 210:21-46. [PMID: 39824582 DOI: 10.1016/bs.pmbts.2024.07.018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/20/2025]
Abstract
Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.
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Affiliation(s)
- D A Ayush Gowda
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea
| | - Girish Birappa
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea
| | - Sripriya Rajkumar
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea
| | - C Bindu Ajaykumar
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea
| | | | - Sammy L Kim
- Department of Biological Science, College of Sang-Huh Life Science, Department of Biological Science, Konkuk University, Seoul, South Korea
| | - Vijai Singh
- Department of Biosciences, School of Science, Indrashil University, Rajpur, Mehsana, Gujarat, India
| | - Aparna Jayachandran
- Fiona Elsey Cancer Research Institute, VIC, Australia; Federation University, VIC, Australia.
| | - Junwon Lee
- Department of Ophthalmology, Institute of Vision Research, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea.
| | - Suresh Ramakrishna
- Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea.
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18
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Feng Q, Li Q, Zhou H, Wang Z, Lin C, Jiang Z, Liu T, Wang D. CRISPR technology in human diseases. MedComm (Beijing) 2024; 5:e672. [PMID: 39081515 PMCID: PMC11286548 DOI: 10.1002/mco2.672] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2023] [Revised: 07/01/2024] [Accepted: 07/01/2024] [Indexed: 08/02/2024] Open
Abstract
Gene editing is a growing gene engineering technique that allows accurate editing of a broad spectrum of gene-regulated diseases to achieve curative treatment and also has the potential to be used as an adjunct to the conventional treatment of diseases. Gene editing technology, mainly based on clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein systems, which is capable of generating genetic modifications in somatic cells, provides a promising new strategy for gene therapy for a wide range of human diseases. Currently, gene editing technology shows great application prospects in a variety of human diseases, not only in therapeutic potential but also in the construction of animal models of human diseases. This paper describes the application of gene editing technology in hematological diseases, solid tumors, immune disorders, ophthalmological diseases, and metabolic diseases; focuses on the therapeutic strategies of gene editing technology in sickle cell disease; provides an overview of the role of gene editing technology in the construction of animal models of human diseases; and discusses the limitations of gene editing technology in the treatment of diseases, which is intended to provide an important reference for the applications of gene editing technology in the human disease.
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Affiliation(s)
- Qiang Feng
- Laboratory Animal CenterCollege of Animal ScienceJilin UniversityChangchunChina
- Research and Development CentreBaicheng Medical CollegeBaichengChina
| | - Qirong Li
- Laboratory Animal CenterCollege of Animal ScienceJilin UniversityChangchunChina
| | - Hengzong Zhou
- Laboratory Animal CenterCollege of Animal ScienceJilin UniversityChangchunChina
| | - Zhan Wang
- Laboratory Animal CenterCollege of Animal ScienceJilin UniversityChangchunChina
| | - Chao Lin
- School of Grain Science and TechnologyJilin Business and Technology CollegeChangchunChina
| | - Ziping Jiang
- Department of Hand and Foot SurgeryThe First Hospital of Jilin UniversityChangchunChina
| | - Tianjia Liu
- Research and Development CentreBaicheng Medical CollegeBaichengChina
| | - Dongxu Wang
- Laboratory Animal CenterCollege of Animal ScienceJilin UniversityChangchunChina
- Department of Hand and Foot SurgeryThe First Hospital of Jilin UniversityChangchunChina
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19
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Irfan M, Majeed H, Iftikhar T, Ravi PK. A review on molecular scissoring with CRISPR/Cas9 genome editing technology. Toxicol Res (Camb) 2024; 13:tfae105. [PMID: 39006883 PMCID: PMC11240166 DOI: 10.1093/toxres/tfae105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Accepted: 07/02/2024] [Indexed: 07/16/2024] Open
Abstract
Genome editing is a technology to make specific changes in the DNA of a cell or an organism. It has significantly altered the landscape of life sciences, facilitating the establishment of exceedingly customized genetic modifications. Among various genome editing technologies, the CRISPR/Cas9 system, a specific endonuclease induces a double stranded DNA break and enabling modifications to the genome, has surfaced as a formidable and adaptable instrument. Its significance cannot be overstated, as it not only allows for the manipulation of genomes in model organisms but also holds great potential for revolutionary advances in medicine, particularly in treating genetic diseases. This review paper explores the remarkable journey of CRISPR/Cas9, its natural function, mechanisms, and transformative impact on genome editing and finally the use of artificial intelligence and other intelligent manufacturing tools used. The introduction provides the background on genome editing, emphasizing the emergence and significance of CRISPR/Cas9. Subsequent sections comprehensively elucidate its natural function, disease modeling, agriculture, and biotechnology, address therapeutic applications, and ongoing clinical trials while also discussing prospects and ethical implications. We summarized the key findings, indicating that CRISPR/Cas9 has empowered the creation of disease-specific animal models. This provides invaluable insights into pathogenic mechanisms and opens new avenues for drug discovery, reaffirming the transformative impact of CRISPR/Cas9 on genome editing. Finally we discussed the importance of continued research and collaboration for comprehensive utilization of the inherent capabilities of this molecular precision tool in shaping forthcoming advancements.
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Affiliation(s)
- Muskan Irfan
- Department of Biotechnology, University of Management and Technology (UMT), Lahore, Sialkot Campus, Sialkot 51310, Pakistan
| | - Hammad Majeed
- Department of Chemistry, University of Management and Technology (UMT), Lahore, Sialkot Campus, Sialkot 51310, Pakistan
| | - Tehreema Iftikhar
- Applied Botany Lab, Department of Botany, Government College University, 54000, Lahore, Pakistan
| | - Pritam Kumar Ravi
- Computer Applications Department, Ganesh Lal Agarwal College, Nilamber-Pitamber University, Jharkhand, 822101, India
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20
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Luo R, Le H, Wu Q, Gong C. Nanoplatform-Based In Vivo Gene Delivery Systems for Cancer Therapy. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2024; 20:e2312153. [PMID: 38441386 DOI: 10.1002/smll.202312153] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Revised: 02/05/2024] [Indexed: 07/26/2024]
Abstract
Gene therapy uses modern molecular biology methods to repair disease-causing genes. As a burgeoning therapeutic, it has been widely applied for cancer therapy. Since 1989, there have been numerous clinical gene therapy cases worldwide. However, a few are successful. The main challenge of clinical gene therapy is the lack of efficient and safe vectors. Although viral vectors show high transfection efficiency, their application is still limited by immune rejection and packaging capacity. Therefore, the development of non-viral vectors is overwhelming. Nanoplatform-based non-viral vectors become a hotspot in gene therapy. The reasons are mainly as follows. 1) Non-viral vectors can be engineered to be uptaken by specific types of cells or tissues, providing effective targeting capability. 2) Non-viral vectors can protect goods that need to be delivered from degradation. 3) Nanoparticles can transport large-sized cargo such as CRISPR/Cas9 plasmids and nucleoprotein complexes. 4) Nanoparticles are highly biosafe, and they are not mutagenic in themselves compared to viral vectors. 5) Nanoparticles are easy to scale preparation, which is conducive to clinical conversion and application. Here, an overview of the categories of nanoplatform-based non-viral gene vectors, the limitations on their development, and their applications in cancer therapy.
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Affiliation(s)
- Rui Luo
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Hao Le
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Qinjie Wu
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Changyang Gong
- Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China
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21
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Kurzawa-Akanbi M, Tzoumas N, Corral-Serrano JC, Guarascio R, Steel DH, Cheetham ME, Armstrong L, Lako M. Pluripotent stem cell-derived models of retinal disease: Elucidating pathogenesis, evaluating novel treatments, and estimating toxicity. Prog Retin Eye Res 2024; 100:101248. [PMID: 38369182 DOI: 10.1016/j.preteyeres.2024.101248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Revised: 02/13/2024] [Accepted: 02/14/2024] [Indexed: 02/20/2024]
Abstract
Blindness poses a growing global challenge, with approximately 26% of cases attributed to degenerative retinal diseases. While gene therapy, optogenetic tools, photosensitive switches, and retinal prostheses offer hope for vision restoration, these high-cost therapies will benefit few patients. Understanding retinal diseases is therefore key to advance effective treatments, requiring in vitro models replicating pathology and allowing quantitative assessments for drug discovery. Pluripotent stem cells (PSCs) provide a unique solution given their limitless supply and ability to differentiate into light-responsive retinal tissues encompassing all cell types. This review focuses on the history and current state of photoreceptor and retinal pigment epithelium (RPE) cell generation from PSCs. We explore the applications of this technology in disease modelling, experimental therapy testing, biomarker identification, and toxicity studies. We consider challenges in scalability, standardisation, and reproducibility, and stress the importance of incorporating vasculature and immune cells into retinal organoids. We advocate for high-throughput automation in data acquisition and analyses and underscore the value of advanced micro-physiological systems that fully capture the interactions between the neural retina, RPE, and choriocapillaris.
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22
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Gao M, He Y, Zhu X, Peng W, Zhou Y, Deng Y, Liao G, Ni W, Li Y, Gao J, Bu H, Yang J, Yang G, Yang Y, Bao J. One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses. Front Cell Dev Biol 2024; 12:1376936. [PMID: 38559814 PMCID: PMC10978582 DOI: 10.3389/fcell.2024.1376936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 02/29/2024] [Indexed: 04/04/2024] Open
Abstract
Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.
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Affiliation(s)
- Mengyu Gao
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - YuTing He
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - XingLong Zhu
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - WanLiu Peng
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - YanYan Zhou
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Yang Deng
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Guangneng Liao
- Experimental Animal Center, West China Hospital, Sichuan University, Chengdu, China
| | - Wei Ni
- Security Department, West China Hospital, Sichuan University, Chengdu, China
| | - Yi Li
- Institute of Respiratory Health, West China Hospital, Sichuan University, Chengdu, China
| | - Jun Gao
- Department of Toxicological Inspection, Sichuan Center for Disease Prevention and Control, Chengdu, China
| | - Hong Bu
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Jiayin Yang
- Laboratory of Liver Transplantation, Frontiers Science Center for Disease-related Molecular, Network, West China Hospital, Sichuan University, Chengdu, China
| | - Guang Yang
- Experimental Animal Center, West China Hospital, Sichuan University, Chengdu, China
| | - Yang Yang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China
| | - Ji Bao
- Department of Pathology, Institute of Clinical Pathology, Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
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23
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De Angeli P, Flores-Tufiño A, Stingl K, Kühlewein L, Roschi E, Wissinger B, Kohl S. Splicing defects and CRISPR-Cas9 correction in isogenic homozygous photoreceptor precursors harboring clustered deep-intronic ABCA4 variants. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102113. [PMID: 38274366 PMCID: PMC10809099 DOI: 10.1016/j.omtn.2023.102113] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Accepted: 12/21/2023] [Indexed: 01/27/2024]
Abstract
Splicing defects from deep-intronic variants significantly contribute to the mutational spectrum in ABCA4-associated inherited retinal diseases, necessitating functional validation for their pathological classification. Typically, minigene assays in HEK293(T) can qualitatively assess splicing defects, yet they often fail to quantitatively reproduce the resulting mis-splicing patterns, leaving uncertainty on severity and pathogenicity. As a potential cellular model derived from patient cells, photoreceptor precursor cells (PPCs) play a pivotal role in assessing the severity of specific splicing mutations. Nevertheless, the accessibility of biosamples is commonly constrained, and their establishment is costly and laborious. In this study, we combined and investigated the use of a minigene assay and isogenic PPCs, as superior qualitative and more accessible cellular models for the assessment of splicing defects. Specifically, we focused on the clustered c.5196+1013A>G, c.5196+1056A>G, and c.5196+1216C>A deep-intronic variants in intron 36 of ABCA4, comparing their resulting (mis)splicing patterns in minigene-transfected cells and isogenic CRISPR-Cas9-knocked-in PPCs harboring these pathogenic variants in homozygous state. Moreover, we demonstrate the successful correction of these three splicing defects in homozygous mutant PPCs using a single pair of guide RNAs to target Cas9 cleavage, thereby identifying an efficient gene editing strategy for therapeutic applications.
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Affiliation(s)
- Pietro De Angeli
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
| | - Arturo Flores-Tufiño
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
| | - Katarina Stingl
- University Eye Hospital, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
| | - Laura Kühlewein
- University Eye Hospital, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
| | - Eleonora Roschi
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
- Wellcome Sanger Institute, Hinxton CB10 1RQ, Saffron Walden, UK
| | - Bernd Wissinger
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
| | - Susanne Kohl
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Clinics Tübingen, Elfriede-Aulhorn-Str. 7, 72076 Tübingen, Germany
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24
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Du X, Butler AG, Chen HY. Cell-cell interaction in the pathogenesis of inherited retinal diseases. Front Cell Dev Biol 2024; 12:1332944. [PMID: 38500685 PMCID: PMC10944940 DOI: 10.3389/fcell.2024.1332944] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Accepted: 02/06/2024] [Indexed: 03/20/2024] Open
Abstract
The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.
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Affiliation(s)
| | | | - Holly Y. Chen
- Department of Cell, Developmental and Integrative Biology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, AL, United States
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25
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Davis DJ, Yeddula SGR. CRISPR Advancements for Human Health. MISSOURI MEDICINE 2024; 121:170-176. [PMID: 38694604 PMCID: PMC11057861] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 05/04/2024]
Abstract
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.
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Affiliation(s)
- Daniel J Davis
- Assistant Director - Animal Modeling Core; Assistant Research Professor - Department of Veterinary Pathobiology; and Comparative Medicine Program Faculty, University of Missouri - Columbia, Columbia, Missouri
| | - Sai Goutham Reddy Yeddula
- PhD candidate in the Department of Animal Sciences, University of Missouri - Columbia, Columbia, Missouri
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26
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Sundaresan Y, Yacoub S, Kodati B, Amankwa CE, Raola A, Zode G. Therapeutic applications of CRISPR/Cas9 gene editing technology for the treatment of ocular diseases. FEBS J 2023; 290:5248-5269. [PMID: 36877952 PMCID: PMC10480348 DOI: 10.1111/febs.16771] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2022] [Revised: 02/04/2023] [Accepted: 03/03/2023] [Indexed: 03/08/2023]
Abstract
Ocular diseases are a highly heterogeneous group of phenotypes, caused by a spectrum of genetic variants and environmental factors that exhibit diverse clinical symptoms. As a result of its anatomical location, structure and immune privilege, the eye is an ideal system to assess and validate novel genetic therapies. Advances in genome editing have revolutionized the field of biomedical science, enabling researchers to understand the biology behind disease mechanisms and allow the treatment of several health conditions, including ocular pathologies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing facilitates efficient and specific genetic modifications in the nucleic acid sequence, resulting in permanent changes at the genomic level. This approach has advantages over other treatment strategies and is promising for the treatment of various genetic and non-genetic ocular conditions. This review provides an overview of the CRISPR/CRISPR-associated protein 9 (Cas9) system and summarizes recent advances in the therapeutic application of CRISPR/Cas9 for the treatment of various ocular pathologies, as well as future challenges.
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Affiliation(s)
| | | | - Bindu Kodati
- Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX 76107
| | - Charles E. Amankwa
- Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX 76107
| | - Akash Raola
- Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX 76107
| | - Gulab Zode
- Department of Pharmacology and Neuroscience, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX 76107
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27
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Zhu X, Xu J, Ling G, Zhang P. Tunable metal-organic frameworks assist in catalyzing DNAzymes with amplification platforms for biomedical applications. Chem Soc Rev 2023; 52:7549-7578. [PMID: 37817667 DOI: 10.1039/d3cs00386h] [Citation(s) in RCA: 24] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/12/2023]
Abstract
Various binding modes of tunable metal organic frameworks (MOFs) and functional DNAzymes (Dzs) synergistically catalyze the emergence of abundant functional nanoplatforms. Given their serial variability in formation, structural designability, and functional controllability, Dzs@MOFs tend to be excellent building blocks for the precise "intelligent" manufacture of functional materials. To present a clear outline of this new field, this review systematically summarizes the progress of Dz integration into MOFs (MOFs@Dzs) through different methods, including various surface infiltration, pore encapsulation, covalent binding, and biomimetic mineralization methods. Atomic-level and time-resolved catalytic mechanisms for biosensing and imaging are made possible by the complex interplay of the distinct molecular structure of Dzs@MOF, conformational flexibility, and dynamic regulation of metal ions. Exploiting the precision of DNAzymes, MOFs@Dzs constructed a combined nanotherapy platform to guide intracellular drug synthesis, photodynamic therapy, catalytic therapy, and immunotherapy to enhance gene therapy in different ways, solving the problems of intracellular delivery inefficiency and insufficient supply of cofactors. MOFs@Dzs nanostructures have become excellent candidates for biosensing, bioimaging, amplification delivery, and targeted cancer gene therapy while emphasizing major advancements and seminal endeavors in the fields of biosensing (nucleic acid, protein, enzyme activity, small molecules, and cancer cells), biological imaging, and targeted cancer gene delivery and gene therapy. Overall, based on the results demonstrated to date, we discuss the challenges that the emerging MOFs@Dzs might encounter in practical future applications and briefly look forward to their bright prospects in other fields.
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Affiliation(s)
- Xiaoguang Zhu
- Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China.
| | - Jiaqi Xu
- Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China.
| | - Guixia Ling
- Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China.
| | - Peng Zhang
- Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China.
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28
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Khlidj Y. What did CRISPR-Cas9 accomplish in its first 10 years? Biochem Med (Zagreb) 2023; 33:030601. [PMID: 37545694 PMCID: PMC10373057 DOI: 10.11613/bm.2023.030601] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Accepted: 07/01/2023] [Indexed: 08/08/2023] Open
Abstract
It's been 10 years now from the debut of clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) era in which gene engineering has never been so accessible, precise and efficient. This technology, like a refined surgical procedure, has offered the ability of removing different types of disease causing mutations and restoring key proteins activity with ease of outperforming the previous resembling methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Additionally, CRISPR-Cas9 systems can systematically introduce genetic sequences to the specific sites in the human genome allowing to stimulate desired functions such as anti-tumoral and anti-infectious faculties. The present brief review provides an updated resume of CRISPR-Cas9's top achievements from its first appearance to the current date focusing on the breakthrough research including in vitro, in vivo and human studies. This enables the evaluation of the previous phase 'the proof-of-concept phase' and marks the beginning of the next phase which will probably bring a spate of clinical trials.
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29
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Gour A, Tibrewal S, Garg A, Vohra M, Ratna R, Sangwan VS. New horizons in aniridia management: Clinical insights and therapeutic advances. Taiwan J Ophthalmol 2023; 13:467-478. [PMID: 38249501 PMCID: PMC10798387 DOI: 10.4103/tjo.tjo-d-23-00140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2023] [Accepted: 10/11/2023] [Indexed: 01/23/2024] Open
Abstract
Congenital aniridia is a rare genetic eye disorder characterized by the complete or partial absence of the iris from birth. Various theories and animal models have been proposed to understand and explain the pathogenesis of aniridia. In the majority of cases, aniridia is caused by a mutation in the PAX6 gene, which affects multiple structures within the eye. Treating these ocular complications is challenging and carries a high risk of side effects. However, emerging approaches for the treatment of aniridia-associated keratopathy, iris abnormalities, cataract abnormalities, and foveal hypoplasia show promise for improved outcomes. Genetic counseling plays a very important role to make informed choices. We also provide an overview of the newer diagnostic and therapeutic approaches such as next generation sequencing, gene therapy, in vivo silencing, and miRNA modulation.
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Affiliation(s)
- Abha Gour
- Department of Cornea and Anterior Segment, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
- Eicher-Shroff Centre for Stem Cell Research, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
| | - Shailaja Tibrewal
- Department of Pediatric Ophthalmology and Strabismus, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
- Department of Ocular Genetics, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
| | - Aastha Garg
- Department of Cornea and Anterior Segment, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
| | - Mehak Vohra
- Eicher-Shroff Centre for Stem Cell Research, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
| | - Ria Ratna
- Department of Ocular Genetics, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
| | - Virender Singh Sangwan
- Department of Cornea and Anterior Segment, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
- Eicher-Shroff Centre for Stem Cell Research, Dr. Shroff’s Charity Eye Hospital, New Delhi, India
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30
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Sahli E, Kiziltunc PB, Idil A. A Report on Children with CEP290 Mutation, Vision Loss, and Developmental Delay. BEYOGLU EYE JOURNAL 2023; 8:226-232. [PMID: 37766766 PMCID: PMC10521126 DOI: 10.14744/bej.2023.37233] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 05/28/2023] [Accepted: 06/24/2023] [Indexed: 09/29/2023]
Abstract
Mutations in CEP290, which encodes a centrosomal protein, cause Joubert syndrome, retinal dystrophy, and several other manifestations. Retinal dystrophy related to CEP290 mutation (Leber's congenital amaurosis type 10) presents with a severe visual impairment from birth, wandering eye movements, and oculodigital reflex. Fundus examination may initially be normal, but varying degrees of retinal pigmentation can be detected over time. This report presents 4 children who were referred to the ophthalmology clinic with a lack of eye contact and the suspicion of low vision. The ophthalmological examination revealed very poor visual function, the vision slightly improved over time, and enophthalmos became evident. There was neuromotor retardation in their history and mutations in the CEP290 gene were revealed in the whole-exome analysis. Both pediatricians and ophthalmologists should be aware of the coincidence between severe vision loss and neuromotor retardation and should refer patients for genetic testing if they suspect it. Genetic diagnosis will enable patients to be followed both neurologically and ophthalmologically and to benefit from rehabilitation opportunities that will contribute to visual and neurological development. It will also allow the family to receive genetic counseling on disease progression and heredity, and to follow ongoing gene therapy studies for mutations in the relevant gene.
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Affiliation(s)
- Esra Sahli
- Department of Ophthalmology, Ankara University, Faculty of Medicine, Ankara, Türkiye
| | | | - Aysun Idil
- Department of Ophthalmology, Ankara University, Faculty of Medicine, Ankara, Türkiye
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31
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Ebrahimi S, Khosravi MA, Raz A, Karimipoor M, Parvizi P. CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications. IRANIAN BIOMEDICAL JOURNAL 2023; 27:219-46. [PMID: 37873636 PMCID: PMC10707817 DOI: 10.61186/ibj.27.5.219] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Accepted: 06/14/2023] [Indexed: 12/17/2023]
Abstract
Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.
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Affiliation(s)
- Sahar Ebrahimi
- Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Mohammad Ali Khosravi
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Abbasali Raz
- Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Morteza Karimipoor
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Parviz Parvizi
- Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran
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32
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Zufiaurre-Seijo M, García-Arumí J, Duarri A. Clinical and Molecular Aspects of C2orf71/PCARE in Retinal Diseases. Int J Mol Sci 2023; 24:10670. [PMID: 37445847 DOI: 10.3390/ijms241310670] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2023] [Revised: 06/20/2023] [Accepted: 06/21/2023] [Indexed: 07/15/2023] Open
Abstract
Mutations in the photoreceptor-specific C2orf71 gene (also known as photoreceptor cilium actin regulator protein PCARE) cause autosomal recessive retinitis pigmentosa type 54 and cone-rod dystrophy. No treatments are available for patients with C2orf71 retinal ciliopathies exhibiting a severe clinical phenotype. Our understanding of the disease process and the role of PCARE in the healthy retina significantly limits our capacity to transfer recent technical developments into viable therapy choices. This study summarizes the current understanding of C2orf71-related retinal diseases, including their clinical manifestations and an unclear genotype-phenotype correlation. It discusses molecular and functional studies on the photoreceptor-specific ciliary PCARE, focusing on the photoreceptor cell and its ciliary axoneme. It is proposed that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane during the development of a new outer segment disk in photoreceptor cells. This review also introduces various cellular and animal models used to model these diseases and provides an overview of potential treatments.
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Affiliation(s)
- Maddalen Zufiaurre-Seijo
- Ophthalmology Research Group, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, 08035 Barcelona, Spain
| | - José García-Arumí
- Ophthalmology Research Group, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, 08035 Barcelona, Spain
| | - Anna Duarri
- Ophthalmology Research Group, Vall d'Hebron Institut de Recerca (VHIR), Vall d'Hebron Hospital Universitari, 08035 Barcelona, Spain
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33
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Starr C, Chen B. Adeno-associated virus mediated gene therapy for neuroprotection of retinal ganglion cells in glaucoma. Vision Res 2023; 206:108196. [PMID: 36812679 PMCID: PMC10085843 DOI: 10.1016/j.visres.2023.108196] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 02/10/2023] [Accepted: 02/10/2023] [Indexed: 02/22/2023]
Abstract
Glaucoma is a group of diseases typically characterized by the degeneration of the optic nerve and is one of the world's leading causes of blindness. Although there is no cure for glaucoma, reducing intraocular pressure is an approved treatment to delay optic nerve degeneration and retinal ganglion cell (RGC) death in most patients. Recent clinical trials have evaluated the safety and efficacy of gene therapy vectors for the treatment of inherited retinal degenerations (IRDs), and the results are promising, generating enthusiasm for the treatment of other retinal diseases. While there have been no reports on successful clinical trials for gene therapy-based neuroprotective treatment of glaucoma, and only a few studies assessing the efficacy of gene therapy vectors for the treatment of Leber hereditary optic neuropathy (LHON), the potential for neuroprotective treatment of glaucoma and other diseases affecting RGCs is still widely recognized. Here, we review recent progress and cover current limitations pertaining to targeting RGCs with adeno-associated virus-based gene therapy for the treatment of glaucoma.
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Affiliation(s)
- Christopher Starr
- Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Optometry and Vision Science, The University of Alabama at Birmingham, Birmingham, AL 35233, USA
| | - Bo Chen
- Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
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34
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Zhou L, Yao S. Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications. MOLECULAR BIOMEDICINE 2023; 4:10. [PMID: 37027099 PMCID: PMC10080534 DOI: 10.1186/s43556-023-00115-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2022] [Accepted: 01/04/2023] [Indexed: 04/08/2023] Open
Abstract
Recently, clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 derived editing tools had significantly improved our ability to make desired changes in the genome. Wild-type Cas9 protein recognizes the target genomic loci and induced local double strand breaks (DSBs) in the guidance of small RNA molecule. In mammalian cells, the DSBs are mainly repaired by endogenous non-homologous end joining (NHEJ) pathway, which is error prone and results in the formation of indels. The indels can be harnessed to interrupt gene coding sequences or regulation elements. The DSBs can also be fixed by homology directed repair (HDR) pathway to introduce desired changes, such as base substitution and fragment insertion, when proper donor templates are provided, albeit in a less efficient manner. Besides making DSBs, Cas9 protein can be mutated to serve as a DNA binding platform to recruit functional modulators to the target loci, performing local transcriptional regulation, epigenetic remolding, base editing or prime editing. These Cas9 derived editing tools, especially base editors and prime editors, can introduce precise changes into the target loci at a single-base resolution and in an efficient and irreversible manner. Such features make these editing tools very promising for therapeutic applications. This review focuses on the evolution and mechanisms of CRISPR-Cas9 derived editing tools and their applications in the field of gene therapy.
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Affiliation(s)
- Lifang Zhou
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu, 610041, Sichuan, China
| | - Shaohua Yao
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu, 610041, Sichuan, China.
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O'Geen H, Beitnere U, Garcia MS, Adhikari A, Cameron DL, Fenton TA, Copping NA, Deng P, Lock S, Halmai JANM, Villegas IJ, Liu J, Wang D, Fink KD, Silverman JL, Segal DJ. Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model. Mol Ther 2023; 31:1088-1105. [PMID: 36641623 PMCID: PMC10124086 DOI: 10.1016/j.ymthe.2023.01.013] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2022] [Revised: 12/19/2022] [Accepted: 01/10/2023] [Indexed: 01/15/2023] Open
Abstract
Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.
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Affiliation(s)
| | | | | | - Anna Adhikari
- MIND Institute, UC Davis Health System, Sacramento, CA, USA; Department of Psychiatry and Behavioral Sciences, UC Davis Health System, Sacramento, CA, USA
| | - David L Cameron
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Timothy A Fenton
- MIND Institute, UC Davis Health System, Sacramento, CA, USA; Department of Psychiatry and Behavioral Sciences, UC Davis Health System, Sacramento, CA, USA
| | - Nycole A Copping
- MIND Institute, UC Davis Health System, Sacramento, CA, USA; Department of Psychiatry and Behavioral Sciences, UC Davis Health System, Sacramento, CA, USA
| | - Peter Deng
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Samantha Lock
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Julian A N M Halmai
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Isaac J Villegas
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Jiajian Liu
- Genome Editing and Novel Modalities (GENM), MilliporeSigma, St. Louis, MO, USA
| | - Danhui Wang
- Genome Editing and Novel Modalities (GENM), MilliporeSigma, St. Louis, MO, USA
| | - Kyle D Fink
- Neurology Department, Stem Cell Program and Gene Therapy Center, UC Davis Health System, Sacramento, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA
| | - Jill L Silverman
- MIND Institute, UC Davis Health System, Sacramento, CA, USA; Department of Psychiatry and Behavioral Sciences, UC Davis Health System, Sacramento, CA, USA
| | - David J Segal
- Genome Center, UC Davis, Davis, CA, USA; Department of Biochemistry and Molecular Medicine, UC Davis, Davis, CA, USA; MIND Institute, UC Davis Health System, Sacramento, CA, USA.
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36
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Ling J, Jenny LA, Zhou A, Tsang SH. Therapeutic Gene Editing in Inherited Retinal Disorders. Cold Spring Harb Perspect Med 2023; 13:a041292. [PMID: 36096547 PMCID: PMC10071418 DOI: 10.1101/cshperspect.a041292] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Since the development of CRISPR/Cas9 gene editing in 2012, therapeutic editing research has produced several phase 1-2a trials. Here we provide an overview of the mechanisms and applications of various gene-editing technologies including adeno-associated virus vectors, lentiviruses, CRISPR/Cas9 systems, base and prime editing, antisense oligonucleotides, short-hairpin RNAs, Cas13, and adenosine deaminase acting on RNA for the treatment of various inherited retinal diseases (IRDs). We outline the various stages of clinical trials using these technologies and the impacts they have made in advancing the practice of medicine.
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Affiliation(s)
- Jinjie Ling
- Columbia University Vagelos College of Physicians and Surgeons, New York, New York 10032, USA
| | - Laura A Jenny
- Jonas Children's Vision Care, and Bernard and Shirley Brown Glaucoma Laboratory, Edward Harkness Eye Institute, Department of Ophthalmology, New York-Presbyterian Hospital, New York, New York 10032, USA
| | - Ashley Zhou
- Columbia University Vagelos College of Physicians and Surgeons, New York, New York 10032, USA
| | - Stephen H Tsang
- Jonas Children's Vision Care, and Bernard and Shirley Brown Glaucoma Laboratory, Edward Harkness Eye Institute, Department of Ophthalmology, New York-Presbyterian Hospital, New York, New York 10032, USA
- Department of Biomedical Engineering, Columbia University, New York, New York 10032, USA
- Columbia Stem Cell Initiative, and Institute of Human Nutrition, Columbia University, New York, New York 10032, USA
- Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA
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37
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Wu M, Li H, Zhang C, Wang Y, Zhang C, Zhang Y, Zhong A, Zhang D, Liu X. Silk-Gel Powered Adenoviral Vector Enables Robust Genome Editing of PD-L1 to Augment Immunotherapy across Multiple Tumor Models. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2206399. [PMID: 36840638 PMCID: PMC10131848 DOI: 10.1002/advs.202206399] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/02/2022] [Revised: 01/16/2023] [Indexed: 06/18/2023]
Abstract
Immune checkpoint blockade based on antibodies has shown great clinical success in patients, but the transitory working manner leads to restricted therapeutic benefits. Herein, a genetically engineered adenovirus is developed as the vector to deliver CRISPR/Cas9 (sgCas9-AdV) to achieve permanent PD-L1 gene editing with efficiency up to 78.7% exemplified in Hepa 1-6 liver cancer cells. Furthermore, the sgCas9-AdV is loaded into hydrogel made by silk fiber (SgCas9-AdV/Gel) for in vivo application. The silk-gel not only promotes local retention of sgCas9-AdV in tumor tissue, but also masks them from host immune system, thus ensuring effectively gene transduction over 9 days. Bearing these advantages, the sgCas9-AdV/Gel inhibits Hepa 1-6 tumor growth with 100% response rate by single-dose injection, through efficient PD-L1 disruption to elicit a T cell-mediated antitumor response. In addition, the sgCas9-AdV/Gel is also successfully extended into other refractory tumors. In CT26 colon tumor characterized by poor response to anti-PD-L1, sgCas9-AdV/Gel is demonstrated to competent and superior anti-PD-L1 antibody to suppress tumor progression. In highly aggressive orthotopic 4T1 mouse breast tumor, such a therapeutic paradigm significantly inhibits primary tumor growth and induces a durable immune response against tumor relapse/metastasis. Thus, this study provides an attractive and universal strategy for immunotherapy.
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Affiliation(s)
- Ming Wu
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- The Liver Center of Fujian ProvinceFujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
- Fujian Provincial Clinical Research Center for Hepatobiliary and Pancreatic TumorsMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
| | - Hao Li
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
| | - Cao Zhang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
| | - Yingchao Wang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- The Liver Center of Fujian ProvinceFujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
- Fujian Provincial Clinical Research Center for Hepatobiliary and Pancreatic TumorsMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
| | - Cuilin Zhang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- The Liver Center of Fujian ProvinceFujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
- Fujian Provincial Clinical Research Center for Hepatobiliary and Pancreatic TumorsMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
| | - Yuting Zhang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
| | - Aoxue Zhong
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
| | - Da Zhang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- The Liver Center of Fujian ProvinceFujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
- Fujian Provincial Clinical Research Center for Hepatobiliary and Pancreatic TumorsMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
| | - Xiaolong Liu
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian ProvinceMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
- The Liver Center of Fujian ProvinceFujian Medical UniversityFuzhou350025P. R. China
- Mengchao Med‐X CenterFuzhou UniversityFuzhou350116P. R. China
- Fujian Provincial Clinical Research Center for Hepatobiliary and Pancreatic TumorsMengchao Hepatobiliary Hospital of Fujian Medical UniversityFuzhou350025P. R. China
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Hu X, Zhang B, Li X, Li M, Wang Y, Dan H, Zhou J, Wei Y, Ge K, Li P, Song Z. The application and progression of CRISPR/Cas9 technology in ophthalmological diseases. Eye (Lond) 2023; 37:607-617. [PMID: 35915232 PMCID: PMC9998618 DOI: 10.1038/s41433-022-02169-1] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 06/07/2022] [Accepted: 06/30/2022] [Indexed: 11/08/2022] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is an adaptive immune defence system that has gradually evolved in bacteria and archaea to combat invading viruses and exogenous DNA. Advances in technology have enabled researchers to enhance their understanding of the immune process in vivo and its potential for use in genome editing. Thus far, applications of CRISPR/Cas9 genome editing technology in ophthalmology have included gene therapy for corneal dystrophy, glaucoma, congenital cataract, Leber's congenital amaurosis, retinitis pigmentosa, Usher syndrome, fundus neovascular disease, proliferative vitreoretinopathy, retinoblastoma and other eye diseases. Additionally, the combination of CRISPR/Cas9 genome editing technology with adeno-associated virus vector and inducible pluripotent stem cells provides further therapeutic avenues for the treatment of eye diseases. Nonetheless, many challenges remain in the development of clinically feasible retinal genome editing therapy. This review discusses the development, as well as mechanism of CRISPR/Cas9 and its applications and challenges in gene therapy for eye diseases.
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Affiliation(s)
- Xumeng Hu
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Beibei Zhang
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Xiaoli Li
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Miao Li
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Yange Wang
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Handong Dan
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Jiamu Zhou
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Yuanmeng Wei
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Keke Ge
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Pan Li
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China
| | - Zongming Song
- Henan Eye Hospital, Henan Eye Institution, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, 450003, Henan, China.
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39
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Ewaisha R, Anderson KS. Immunogenicity of CRISPR therapeutics-Critical considerations for clinical translation. Front Bioeng Biotechnol 2023; 11:1138596. [PMID: 36873375 PMCID: PMC9978118 DOI: 10.3389/fbioe.2023.1138596] [Citation(s) in RCA: 35] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Accepted: 02/06/2023] [Indexed: 02/18/2023] Open
Abstract
CRISPR offers new hope for many patients and promises to transform the way we think of future therapies. Ensuring safety of CRISPR therapeutics is a top priority for clinical translation and specific recommendations have been recently released by the FDA. Rapid progress in the preclinical and clinical development of CRISPR therapeutics leverages years of experience with gene therapy successes and failures. Adverse events due to immunogenicity have been a major setback that has impacted the field of gene therapy. As several in vivo CRISPR clinical trials make progress, the challenge of immunogenicity remains a significant roadblock to the clinical availability and utility of CRISPR therapeutics. In this review, we examine what is currently known about the immunogenicity of CRISPR therapeutics and discuss several considerations to mitigate immunogenicity for the design of safe and clinically translatable CRISPR therapeutics.
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Affiliation(s)
- Radwa Ewaisha
- Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
- Department of Microbiology and Immunology, School of Pharmacy, Newgiza University, Newgiza, Egypt
| | - Karen S. Anderson
- Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ, United States
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40
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Sellers DL, Lee K, Murthy N, Pun SH. TAxI-peptide targeted Cas12a ribonuclease protein nanoformulations increase genome editing in hippocampal neurons. J Control Release 2023; 354:188-195. [PMID: 36596342 PMCID: PMC9975068 DOI: 10.1016/j.jconrel.2022.12.057] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2022] [Revised: 12/19/2022] [Accepted: 12/28/2022] [Indexed: 01/05/2023]
Abstract
Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.
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Affiliation(s)
- Drew L Sellers
- Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, United States; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, United States.
| | - Kunwoo Lee
- GenEdit Inc., Berkeley, CA, United States
| | - Niren Murthy
- Department of Bioengineering, University of California, Berkeley, CA, United States
| | - Suzie H Pun
- Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington, Seattle, WA 98195, United States.
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41
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Chen H, Durinck S, Patel H, Foreman O, Mesh K, Eastham J, Caothien R, Newman RJ, Roose-Girma M, Darmanis S, Warming S, Lattanzi A, Liang Y, Haley B. Population-wide gene disruption in the murine lung epithelium via AAV-mediated delivery of CRISPR-Cas9 components. MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT 2022; 27:431-449. [DOI: 10.1016/j.omtm.2022.10.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 10/27/2022] [Indexed: 11/13/2022]
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CRISPR-Based Tools for Fighting Rare Diseases. LIFE (BASEL, SWITZERLAND) 2022; 12:life12121968. [PMID: 36556333 PMCID: PMC9787644 DOI: 10.3390/life12121968] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 11/15/2022] [Accepted: 11/16/2022] [Indexed: 11/26/2022]
Abstract
Rare diseases affect the life of a tremendous number of people globally. The CRISPR-Cas system emerged as a powerful genome engineering tool and has facilitated the comprehension of the mechanism and development of therapies for rare diseases. This review focuses on current efforts to develop the CRISPR-based toolbox for various rare disease therapy applications and compares the pros and cons of different tools and delivery methods. We further discuss the therapeutic applications of CRISPR-based tools for fighting different rare diseases.
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43
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Huang J, Zhou Y, Li J, Lu A, Liang C. CRISPR/Cas systems: Delivery and application in gene therapy. Front Bioeng Biotechnol 2022; 10:942325. [PMID: 36483767 PMCID: PMC9723151 DOI: 10.3389/fbioe.2022.942325] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2022] [Accepted: 11/04/2022] [Indexed: 10/17/2023] Open
Abstract
The CRISPR/Cas systems in prokaryotes such as bacteria and archaea are the adaptive immune system to prevent infection from viruses, phages, or other foreign substances. When viruses or phages first invade the bacteria, Cas proteins recognize and cut the DNA from viruses or phages into short fragments that will be integrated into the CRISPR array. Once bacteria are invaded again, the modified CRISPR and Cas proteins react quickly to cut DNA at the specified target location, protecting the host. Due to its high efficiency, versatility, and simplicity, the CRISPR/Cas system has become one of the most popular gene editing technologies. In this review, we briefly introduce the CRISPR/Cas systems, focus on several delivery methods including physical delivery, viral vector delivery, and non-viral vector delivery, and the applications of disease therapy. Finally, some problems in CRISPR/Cas9 technology have been proposed, such as the off-target effects, the efficiency of DNA repair mechanisms, and delivery of CRISPR/Cas system safely and efficiently to the target location.
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Affiliation(s)
- Jie Huang
- Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
| | - Yitong Zhou
- Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
| | - Jie Li
- Department of Laboratory Medicine, Peking University Shenzhen Hospital, Shenzhen, China
| | - Aiping Lu
- Institute of Integrated Bioinfomedicine and Translational Science (IBTS), School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China
- Institute of Arthritis Research in Integrative Medicine, Shanghai Academy of Traditional Chinese Medicine, Shanghai, China
- Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research, Guangzhou, China
| | - Chao Liang
- Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
- Institute of Integrated Bioinfomedicine and Translational Science (IBTS), School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China
- State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
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44
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Çerçi B, Uzay IA, Kara MK, Dinçer P. Clinical trials and promising preclinical applications of CRISPR/Cas gene editing. Life Sci 2022; 312:121204. [PMID: 36403643 DOI: 10.1016/j.lfs.2022.121204] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Revised: 11/03/2022] [Accepted: 11/14/2022] [Indexed: 11/18/2022]
Abstract
Treatment of genetic disorders by genomic manipulation has been the unreachable goal of researchers for many decades. Although our understanding of the genetic basis of genetic diseases has advanced tremendously in the last few decades, the tools developed for genomic editing were not efficient and practical for their use in the clinical setting until now. The recent advancements in the research of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems offered an easy and efficient way to edit the genome and accelerated the research on their potential use in the treatment of genetic disorders. In this review, we summarize the clinical trials that evaluate the CRISPR/Cas systems for treating different genetic diseases and highlight promising preclinical research on CRISPR/Cas mediated treatment of a great diversity of genetic disorders. Ultimately, we discuss the future of CRISPR/Cas mediated genome editing in genetic diseases.
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Affiliation(s)
- Barış Çerçi
- Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey.
| | - Ihsan Alp Uzay
- Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey
| | | | - Pervin Dinçer
- Department of Medical Biology, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey
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45
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The retinal pigmentation pathway in human albinism: Not so black and white. Prog Retin Eye Res 2022; 91:101091. [PMID: 35729001 DOI: 10.1016/j.preteyeres.2022.101091] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 05/24/2022] [Accepted: 05/26/2022] [Indexed: 12/16/2022]
Abstract
Albinism is a pigment disorder affecting eye, skin and/or hair. Patients usually have decreased melanin in affected tissues and suffer from severe visual abnormalities, including foveal hypoplasia and chiasmal misrouting. Combining our data with those of the literature, we propose a single functional genetic retinal signalling pathway that includes all 22 currently known human albinism disease genes. We hypothesise that defects affecting the genesis or function of different intra-cellular organelles, including melanosomes, cause syndromic forms of albinism (Hermansky-Pudlak (HPS) and Chediak-Higashi syndrome (CHS)). We put forward that specific melanosome impairments cause different forms of oculocutaneous albinism (OCA1-8). Further, we incorporate GPR143 that has been implicated in ocular albinism (OA1), characterised by a phenotype limited to the eye. Finally, we include the SLC38A8-associated disorder FHONDA that causes an even more restricted "albinism-related" ocular phenotype with foveal hypoplasia and chiasmal misrouting but without pigmentation defects. We propose the following retinal pigmentation pathway, with increasingly specific genetic and cellular defects causing an increasingly specific ocular phenotype: (HPS1-11/CHS: syndromic forms of albinism)-(OCA1-8: OCA)-(GPR143: OA1)-(SLC38A8: FHONDA). Beyond disease genes involvement, we also evaluate a range of (candidate) regulatory and signalling mechanisms affecting the activity of the pathway in retinal development, retinal pigmentation and albinism. We further suggest that the proposed pigmentation pathway is also involved in other retinal disorders, such as age-related macular degeneration. The hypotheses put forward in this report provide a framework for further systematic studies in albinism and melanin pigmentation disorders.
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46
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Escobar M, Li J, Patel A, Liu S, Xu Q, Hilton IB. Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells. ACS Synth Biol 2022; 11:3239-3250. [PMID: 36162812 PMCID: PMC9594343 DOI: 10.1021/acssynbio.2c00156] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Indexed: 01/24/2023]
Abstract
CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.
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Affiliation(s)
- Mario Escobar
- Department
of BioSciences, Rice University, Houston, Texas 77005, United States
| | - Jing Li
- Department
of Bioengineering, Rice University, Houston, Texas 77005, United States
| | - Aditi Patel
- Department
of BioSciences, Rice University, Houston, Texas 77005, United States
| | - Shizhe Liu
- Department
of BioSciences, Rice University, Houston, Texas 77005, United States
| | - Qi Xu
- Department
of Bioengineering, Rice University, Houston, Texas 77005, United States
| | - Isaac B. Hilton
- Department
of BioSciences, Rice University, Houston, Texas 77005, United States
- Department
of Bioengineering, Rice University, Houston, Texas 77005, United States
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Altay HY, Ozdemir F, Afghah F, Kilinc Z, Ahmadian M, Tschopp M, Agca C. Gene regulatory and gene editing tools and their applications for retinal diseases and neuroprotection: From proof-of-concept to clinical trial. Front Neurosci 2022; 16:924917. [PMID: 36340792 PMCID: PMC9630553 DOI: 10.3389/fnins.2022.924917] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2022] [Accepted: 09/26/2022] [Indexed: 09/11/2023] Open
Abstract
Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.
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Affiliation(s)
- Halit Yusuf Altay
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
| | - Fatma Ozdemir
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
| | - Ferdows Afghah
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
| | - Zeynep Kilinc
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
| | - Mehri Ahmadian
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
| | - Markus Tschopp
- Department of Ophthalmology, Cantonal Hospital Aarau, Aarau, Switzerland
- Department of Ophthalmology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
| | - Cavit Agca
- Molecular Biology, Genetics and Bioengineering Program, Sabanci University, Istanbul, Turkey
- Nanotechnology Research and Application Center (SUNUM), Sabanci University, Istanbul, Turkey
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48
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Li R, Wang Q, She K, Lu F, Yang Y. CRISPR/Cas systems usher in a new era of disease treatment and diagnosis. MOLECULAR BIOMEDICINE 2022; 3:31. [PMID: 36239875 PMCID: PMC9560888 DOI: 10.1186/s43556-022-00095-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Accepted: 09/27/2022] [Indexed: 11/21/2022] Open
Abstract
The discovery and development of the CRISPR/Cas system is a milestone in precise medicine. CRISPR/Cas nucleases, base-editing (BE) and prime-editing (PE) are three genome editing technologies derived from CRISPR/Cas. In recent years, CRISPR-based genome editing technologies have created immense therapeutic potential with safe and efficient viral or non-viral delivery systems. Significant progress has been made in applying genome editing strategies to modify T cells and hematopoietic stem cells (HSCs) ex vivo and to treat a wide variety of diseases and disorders in vivo. Nevertheless, the clinical translation of this unique technology still faces many challenges, especially targeting, safety and delivery issues, which require further improvement and optimization. In addition, with the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), CRISPR-based molecular diagnosis has attracted extensive attention. Growing from the specific set of molecular biological discoveries to several active clinical trials, CRISPR/Cas systems offer the opportunity to create a cost-effective, portable and point-of-care diagnosis through nucleic acid screening of diseases. In this review, we describe the development, mechanisms and delivery systems of CRISPR-based genome editing and focus on clinical and preclinical studies of therapeutic CRISPR genome editing in disease treatment as well as its application prospects in therapeutics and molecular detection.
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Affiliation(s)
- Ruiting Li
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China
| | - Qin Wang
- School of Pharmacy, Southwest Minzu University, Chengdu, 610225, Sichuan, China
| | - Kaiqin She
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Fang Lu
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Yang Yang
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, 610041, Sichuan, China.
- Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
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Jo DH, Bae S, Kim HH, Kim JS, Kim JH. In vivo application of base and prime editing to treat inherited retinal diseases. Prog Retin Eye Res 2022; 94:101132. [PMID: 36241547 DOI: 10.1016/j.preteyeres.2022.101132] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 09/19/2022] [Accepted: 09/28/2022] [Indexed: 11/05/2022]
Abstract
Inherited retinal diseases (IRDs) are vision-threatening retinal disorders caused by pathogenic variants of genes related to visual functions. Genomic analyses in patients with IRDs have revealed pathogenic variants which affect vision. However, treatment options for IRDs are limited to nutritional supplements regardless of genetic variants or gene-targeting approaches based on antisense oligonucleotides and adeno-associated virus vectors limited to targeting few genes. Genome editing, particularly that involving clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technologies, can correct pathogenic variants and provide additional treatment opportunities. Recently developed base and prime editing platforms based on CRISPR-Cas9 technologies are promising for therapeutic genome editing because they do not employ double-stranded breaks (DSBs), which are associated with P53 activation, large deletions, and chromosomal translocations. Instead, using attached deaminases and reverse transcriptases, base and prime editing efficiently induces specific base substitutions and intended genetic changes (substitutions, deletions, or insertions), respectively, without DSBs. In this review, we will discuss the recent in vivo application of CRISPR-Cas9 technologies, focusing on base and prime editing, in animal models of IRDs.
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Burgess FR, Hall HN, Megaw R. Emerging Gene Manipulation Strategies for the Treatment of Monogenic Eye Disease. Asia Pac J Ophthalmol (Phila) 2022; 11:380-391. [PMID: 36041151 DOI: 10.1097/apo.0000000000000545] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2022] [Accepted: 05/27/2022] [Indexed: 12/15/2022] Open
Abstract
Genetic eye diseases, representing a wide spectrum of simple and complex conditions, are one of the leading causes of visual loss in children and working adults, and progress in the field has led to changes in disease investigation, diagnosis, and management. The past 15 years have seen the emergence of novel therapies for these previously untreatable conditions to the extent that we now have a licensed therapy for one form of genetic eye disease and many more in clinical trial. This is a systematic review of published and ongoing clinical trials of gene therapies for monogenic eye diseases. Databases of clinical trials and the published literature were searched for interventional studies of gene therapies for eye diseases. Standard methodological procedures were used to assess the relevance of search results. A total of 59 registered clinical trials are referenced, showing the significant level of interest in the potential for translation of these therapies from bench to bedside. The breadth of therapy design is encouraging, providing multiple possible therapeutic mechanisms. Some fundamental questions regarding gene therapy for genetic eye diseases remain, such as optimal dosing, the relative benefits of adeno-associated virus (AAV)-packaging and the potential for a significant inflammatory response to the therapy itself. As a result, despite the promise of the eye as a target, it has proven difficult to deliver clinically effective gene therapies to the eye. Despite setbacks, the licensing of Luxturna (voretigene neparvovec, Novartis) for the treatment of RPE65-mediated Leber congenital amaurosis (LCA) is a major advance in efforts to treat these rare, but devastating, causes of visual loss.
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Affiliation(s)
- Frederick R Burgess
- Princess Alexandra Eye Pavilion, NHS Lothian, UK
- Ophthalmology Department, School of Medicine, University of St Andrews, UK
| | - Hildegard Nikki Hall
- Princess Alexandra Eye Pavilion, NHS Lothian, UK
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, UK
| | - Roly Megaw
- Princess Alexandra Eye Pavilion, NHS Lothian, UK
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, UK
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